Redox control/monitoring platform for high throughput screening/drug discovery applications

ABSTRACT

The invention is a redox control and monitoring platform that is to be used in conduction with another detection scheme. The platform includes a portion of an electrochemical control. The electrochemical control can be operated to control and measure the redox environment of a sample. The electrochemical control can be provided in a multiplicity of test regions to allow high throughput analysis of a multiplicity of samples. The present method and system allows the determination of the effect of the change in redox environment on the binding or other activity of the species in the sample that is directly affected by the redox environment.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. ProvisionalApplication Ser. No. 60/234,477, filed Sep. 22, 2000.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to a redox control and monitoringplatform and, more particularly, to a redox control and monitoringplatform that is to be used in conjunction with another detector duringhigh throughput screening and drug discovery applications.

[0003] A great number of studies have demonstrated the importance of theredox environment in the regulation of a number of cellular functions inboth normal and diseased states. Such processes include those thatinvolve redox active proteins and enzymes, free radical damage, andoxidative stress. The redox environment can influence both catalysis andbinding affinity. Additionally, transcription of DNA into mRNA, thetranslation of mRNA into proteins, and the rate of transport ofglutamate across the nerve synapses have all been shown to depend on theredox environment. The cellular redox environment has also beenimplicated in the modulation of more complex cellular events such asproliferation and apotosis, and the specific redox environment can alsoactivate certain drugs. It is apparent that the redox environment of atarget molecule or species can have a great affect on the efficacy of aparticular drug.

[0004] Researchers often use high throughput systems to analyze a largelibrary of compounds that could have a desired activity. An example of asystem useful for high throughput screening and assay is shown in U.S.Pat. No. 6,238,869 to Kris, et al, which is hereby incorporated byreference. Such systems allow researchers to quickly test a huge numberof compounds and discard those that do not show the desired activity orquality. Such systems are especially useful in the drug discoveryprocess because large scale testing of a series of compounds can beaccomplished quickly and relatively cheaply. Only those compounds thatshow desired activity are tested further. Without high throughputtechnology, the screening of such a large number of compounds would bevirtually impossible.

[0005] Current high throughput drug screening discovery processes do notprovide for the measurement of the redox environment or the activecontrol of the redox environment of a target. Much of the current highthroughput technology relies on spectroscopic, especially fluorescent,methods to provide information about how or if a particular compound isreacting with a target. However, researchers cannot generally make suchmeasurements while actively controlling the redox environment.

[0006] Many methods do exist to measure the redox environment. Anexample of such a system is shown in U.S. Pat. No. 4,963,815 to Hafeman.However, such a system does not provide for active control of the redoxenvironment of the sample. Additionally, the systems do not provideother data other than electrical measurements, such as spectroscopicmeasurements. The systems also are not adapted for use in a highthroughput process.

[0007] Electrochemistry and spectroscopy have been combined to performvarious studies. Heineman, Spectro-electro-chemistry: Combination ofOptical and Electrochemical Techniques for Studies of Redox Chemistry,Anal. Chem. 1978, 50, 390-402; Asanov et al., Heteroenergetics of BovineSerum Albumin Adsorption from Good Solvents Related to CrystallizationConditions, J. of Colloid and Interface Science 1997, 191, 222-235;Johnson et al., Potential-Dependent Enzymatic Activity in an EnzymeThin-Layer Cell, Anal. Chem 1982, 54, 1377-1383. The thin-layerspectrochemical methods are often used to characterize the fundamentalsof electron transfer between and within redox active enzymes and otherbiomolecules. The change in redox potential changes the ratio of theredox forms of the enzymes that the spectroscopic technique is generallymeasuring. However, these methods cannot be generally performed underconditions where assays independent of the redox potential can beperformed. When independent assays have been performed, they involvedimmobilized biomolecules on a surface or within a membrane. It is knownthat this immobilization can alter the biological activity and thenature and extent of their interaction with proteins or other potentialbinding partners such as drugs. Additionally, these methods are notgenerally applicable to a wide variety of systems and sample types.

[0008] Accordingly, there is a need for a versatile system that canprovide measurement and control of the redox environment that isindependent of other assays that can be performed on the sample,especially in high throughput screening.

SUMMARY OF THE INVENTION

[0009] This need is met by the present invention wherein a system andmethod for the control and measurement of the redox environment isdisclosed. This system can be combined with other existing detectionsystems to provide information about how a species reacts or interactswith other species under a controlled set of redox conditions. Thissystem and method are especially useful for high throughput analysis anddrug discovery.

[0010] In accordance with one embodiment of the present invention amethod for analyzing a sample is provided. The method comprisesproviding an electrochemical control for the redox environment of thesample in a test region and providing a detection scheme consisting ofat least one of the following: electrochemical, spectroscopic,radioassay, or magnetic field measurement. The test region may be anystructure that can hold a sample and allow the detection scheme to beperformed such as a beaker, a test tube, a microplate or other reactionchamber. The electrochemical control is operated to measure and controlthe redox potential of the sample, and the detector is operated toanalyze the sample. The electrochemical control preferably has at leasttwo electrodes.

[0011] The electrochemical detection scheme can be amperometry,voltammetry, capacitance or impedance. Preferred is a spectroscopicdetection scheme such as fluorescence, absorbance, infra red,phosphorescence, chemiluminescence, electroluminescence, Raman, electronspin resonance or refractive index.

[0012] In an alternative embodiment, the electrochemical control can beprovided in a multiplicity of test regions. The test regions can be thewells of a microplate. For example, a ninety-six well microplate can beused. The electrochemical controls can be operated separately to providea different redox environment in each of the wells. A different samplecan be placed in each of the wells of the microplate, and theelectrochemical control and detection scheme can independently controland analyze each of the samples in the microplate wells. The electrodescan be provided in the microplate by mounting the controls on theprotrusions of a second plate. The second plate is placed over themicroplate so that the protrusions fit into the wells of the microplate.The protrusions may be conical or truncated cones.

[0013] This embodiment is particularly suited for high throughputscreening. Combinatorial chemistry can be used to generate a largenumber of chemical compounds targeted to a variety of applications suchas drug discovery or superconductive ceramics. High throughput screeningis then used to sort through the enormous number of candidates in orderto identify those that have the desired property. The assay that is usedin the high throughput screening protocol is designed specifically forthe application for which the candidate compounds are intended. Theelectrochemical control can be operated to provide a different redoxenvironment in each well and thus provide additional information aboutthe interaction of the candidates with the target of the sample underspecific redox conditions.

[0014] In accordance with another embodiment of the present invention astructure is provided for analyzing a sample. The structure consists ofa test region, an electrochemical control for the redox environment ofthe sample and a detection scheme. The detection scheme may be anelectrochemical, spectroscopic, radio assay or magnetic fieldmeasurement detector. The electrochemical control is a system that hasat least two electrodes.

[0015] In a preferred embodiment of the invention, the electrochemicalcontrol is provided in a multiplicity of test regions. The test regionsmay be wells in a microplate. The electrochemical control can beseparately operated to provide a different redox environment in eachwell of the mircroplate. The electrodes are provided in the wells of themicroplate by mounting them on the protrusions of a second plate. Theprotrusions fit into the wells of the microplate. This arrangementallows the detector of the detection scheme access to the sample fromthe bottom of the microplate. The electrodes may be operated prior toand/or during the operation of the detector to allow analysis of thesample under a controlled redox environment.

[0016] In another embodiment of the invention a method of highthroughput drug screening is provided. The method comprises providing asurface comprising a plurality of test regions, providing anelectrochemical control for the redox environment of the test region ineach test region, providing a detection scheme that is selected from atleast one of the following: electrochemical, spectroscopic, radio assayand magnetic field measurement, adding at least one target molecule orspecies to each of the test regions, and adding at least one samplecontaining a drug candidate to be tested to each test region. Theelectrochemical control is operated separately for each test region. Thedetector is subsequently operated to identify if interaction hasoccurred between the target and the sample.

[0017] The electrochemical detection scheme can be amperometry,voltammetry, capacitance or impedance. The spectroscopic detectionscheme can be a fluorescence, absorbance, infra red, phosphorescence,chemiluminescence, electroluminescence, Raman, electron spin resonanceor refractive index.

[0018] The test regions may be a multiplicity of wells in a microplate.A portion of electrochemical control may be contained on a plate withprotrusions wherein the electrodes are deposisted on the protrusions ofthe plate. The plate is placed over the microplate so that theprotrusions of the plate fit into the wells of the microplate. Theprotrusions may be conical or truncated cones.

[0019] In yet another embodiment of the present invention, a method forperforming high throughput assays of non-biological samples is provided.The method comprises providing a surface comprising a plurality of testregions, providing an electrochemical control for the redox environmentof the test region in each test region, providing a detection methodthat is selected from at least one of the following: electrochemical,spectroscopic, radio assay and magnetic field measurement, adding atleast one target molecule or species to each of the test regions, andadding at least one sample containing a species to be tested to eachtest region. The electrochemical control can be operated separately foreach test region. The detector of the detection scheme is subsequentlyoperated to identify if interaction has occurred between the target andthe sample. This method allows the species that shows the desiredactivity under the selected redox conditions to be more easilyidentified. This method could be useful when inorganic compounds orpolymers are synthesized using combinatorial methods as candidates for avariety of applications including superconductive ceramics or conductivepolymers.

[0020] Accordingly, it is an object of the present invention to providean electrochemical control for the redox environment of a sample that iscoupled with an independent detection method. A further object of thepresent invention is to provide a method and system for providingseparate control of the redox environment of a multiplicity of samplesfor increased throughput analysis. Other objects of the presentinvention will be apparent in light of the description of the inventionembodied herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021]FIG. 1 is a cross-section side view of a portion of theelectrochemical control and the test regions showing the top plate andbottom plate separately.

[0022]FIG. 2 is cross-section side view of the mated top plate andbottom plate with conical wells.

[0023]FIG. 3 is a bottom view of the top plate.

[0024]FIG. 4 is a cross-section side view of another embodiment of aportion of the electrochemical control and the test regions showing thetop and bottom plate separately.

[0025]FIG. 5 is a block diagram of the preferred embodiment.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Referring initially to FIGS. 1 and 2, a particular embodiment ofthe electrochemical control assembly of the present invention isdescribed in detail. A control assembly 2 according to the presentinvention includes a bottom plate 30 with sample wells 32, a top plate10 with protrusions 12, and electrodes 14 a, 16, 18 disposed on theprotrusions 12.

[0027] The top plate 10 is configured so that protrusions 12 containingthe electrodes 14 a, 16, 18 fit into the sample wells 32 of the bottomplate 30 when the top plate 10 is placed over the bottom plate 30. Thisconfiguration forms a test region 50. Each of the sample wells 32 canhold a separate sample, and each of the electrode arrangements 14 a, 16,18 on the protrusions 12 can be operated to separately control ormeasure the redox environment of each of the sample wells 32.

[0028] The top plate 10 can be a reusable plate made out of plastic orceramic. The bottom plate 30 can be made of transparent plastic oranother transparent material, and the bottom plate 30 maybe disposable.Referring to FIG. 3, the electrodes 14 a, 16, 18 are connected toelectrical traces 24. The electrodes 14 a, 16, 18 and electrical traces24 are applied onto outer conical protrusions 12 of the top plate 10.The electrodes 14 a, 16, 18 and traces 24 may be applied by sputteringor vacuum deposition. The electrical traces 24 are extended to the outeredge of the top plate 10 where an electrical connection can be madethrough an edge connector (not shown).

[0029] Referring generally to FIGS. 1, 3 and 5, the traces 24 may becoupled to a multi-channel potentiostat, represented by 44. Each set ofelectrodes 14 a, 16, 18 can be coupled to a separate potentiostat 44 viathe traces 24 to allow separate control of the redox environment in eachwell 32. However, all of the electrodes 14 a, 16, 18 could be coupled toa single potentiostat 44 to provide more limited control of the redoxenvironment in each well 32. The potentiostat 44 or potentiostats 44 maybe monitored or controlled with a computer or microprocessor 46. Theelectrochemical control 48 is made up of the electrodes 14 a, 16, 18 onthe top plate 10, the traces 24, the potentiostat 44 or potentiostats 44and the processor 46.

[0030] Referring to FIGS. 1 and 2, three electrodes 14 a, 16, 18 aregenerally provided on each protrusion 12. The working electrode 14 a isthe electrode at which the potential is controlled via the potentiostat.The reference electrode 16 is used as a potentiometric probe in order tomaintain the potential of the working electrode 14 a at a pre-selectedvalue relative to the working electrode 14 a. The auxiliary electrode 18completes the circuit and allows current to flow through the test region50 contained within the sample well 32. A two electrode system couldalso be used. In the two electrode system, potential would be controlledvia a working electrode. A second electrode, the counter electrode,would serve to complete the circuit and act as a reference electrode.However, a three electrode 14 a, 16, 18 system is preferred.

[0031] Referring to FIGS. 2 and 4, the sample is first loaded into thewells 32, 36 of the bottom plate 30, and the top plate 10 is installed.The samples are contained in the thin layer 50 formed by the outerprotrusions 12, 22 of the top plate 10 and the inner surfaces of thesample wells 32, 36 in the bottom plate 30. The thin layer serves as thetest region 50. This arrangement provides a high working electrodesurface area to sample volume ratio. The conical shaped protrusions 12of the embodiment in FIG. 2 prevent bubbles from being trapped in thethin layer 50 after assembly. Rapid equilibration upon changing thepotential on the working electrode 14 a is achieved because thediffusion path to the electrode is minimized. The top plate 10 couldalternatively have protrusions 22 in the shape of a truncated cone 22,as shown in FIG. 4. This could enhance the optical properties of thewells 36 and, thus, provide better excitation and detection.

[0032] Mediator titrants are provided in each of the sample wells 32, 36when the sample includes a larger molecule. Mediator titrants arerelatively small redox active compounds that are used to shuttleelectrons between larger molecules and the electrode. Larger molecules,including many biocomponents, cannot directly exchange electrons at thesurface of the electrode because of their size. Thus, mediator titrantsare used to couple the electrode potential 14 a to the solutionpotential and serve as an electrochemically generated redox titrant toreduce and oxidize the large molecules.

[0033] The mediator titrants useful in the present invention include,but are not limited to: (1) organic molecules such as 4,4′-bipyridine,menadione, menadiol and 4-mercaptopyridine; (2) macrocyles and chelatingligands of transition metals; (3) ferrocene, ferricinium, hydroquinones,quinines; (4) reducible and oxidizable components of organic salts; (5)cobaltcenes and the hexa- and octacyanides of molybdenum, tungsten, andiron; and (6) the trisbypyridyl and hexamine complexes of transitionmetals.

[0034] Referring to FIGS. 2 and 5, the bottom plate 30 is mounted on afixture such that the wells 32 in the bottom plate 30 are in alignmentwith a detection scheme 42. The detection scheme could be a series ofoptical sources and detectors that are mounted below the bottom plate30. Optical excitation can thus be applied and emission can be measuredusing this assembly 2 in conjunction with independent redox controlprovided by the electrochemical control 48 within the sample wells 32.An example of a suitable optical source and detector is contained inU.S. Pat. No. 6,246,046, which is hereby incorporated by references, andwhich discloses a method and apparatus for providing an excitationsource and detector on one side of a micro target.

[0035] The method and system of the present invention do not generallyinvolve the immobilization of biomolecules on the surface of the wells32, 36 or protrusions 12, 22 because this is a bulk solution method.This is advantageous because it is known that immobilization ofbiocomponents can alter their biological activity and the nature andextent of their interaction with proteins or other binding partners suchas drugs. Additionally, there should be no interference from surfaceactivity or interactions. The present invention avoids these problemsassociated with immobilizing the biomolecule on the surface of the wellsand, thus, presents a more accurate picture of how a particular targetreacts.

[0036] Additionally, the method and system of the present inventionallow an assay of the sample that is only indirectly dependent on thechange in redox environment. The present method allows the determinationof the effect of the change in redox environment on the binding or otheractivity of the species that is directly affected by the redoxenvironment. Other methods that utilize redox control and some form ofdetection are generally measuring a change that is directly dependent onthe redox environment. The present invention provides an independentassay that can occur simultaneously with active redox control.

[0037] FIGS. 1-4 show an eight well 32, 36 and protrusion 12, 22assembly 2. Each assembly 2 could have an increased number of wells 32,36 and protrusions 12, 22. For example, a ninety-six well bottom plateis one preferred size because it is currently the standard size for highthroughput assay systems. The well 32, 36 size can be varied so that alarger number of wells 32, 36 can fit onto a plate 30 of the same size.A bottom plate 30 with a large number of wells 32, 36 increases thethroughput of the analysis and results in savings in time and resources.

[0038] It will be obvious to those skilled in the art that variouschanges may be made without departing from the scope of the inventionwhich is not to be considered limited to what is described in thespecification.

What is claimed is:
 1. A method of performing an analysis of a samplecomprising: providing a sample in a test region; providing anelectrochemical control for the redox environment of the sample;operating the electrochemical control to control the redox environmentof the sample; and analyzing the sample using a detection schemeselected from at least one of the following: electrochemical;spectroscopic; radioassay; or magnetic field measurement.
 2. The methodas claimed in claim 1 wherein the electrochemical control has at leasttwo electrodes.
 3. The method as claimed in claim 1 wherein theelectrochemical control has three electrodes wherein the first electrodeis an auxiliary electrode, the second electrode is a referenceelectrode, and the third electrode is a working electrode.
 4. The methodas claimed in claim 1 wherein the electrochemical detection schemecomprises amperometry, voltammetry, capacitance, or impedance.
 5. Themethod as claimed in (claim 1 wherein the spectroscopic detection schemecomprises fluorescence, absorbance, infra red, phosphorescence,chemiluminescence, electroluminescence, Raman, electron spin resonance,or refractive index.
 6. The method as claimed in claim 1 wherein amediator titrant is added to the sample.
 7. The method as claimed inclaim 1 wherein the electrochemical control is provided to control eachof a multiplicity of test regions.
 8. The method as claimed in claim 7wherein the test regions are each of a multiplicity of wells in amicroplate.
 9. The method as claimed in claim 8 wherein theelectrochemical control may be operated to separately control the redoxenvironment of each well in a microplate.
 10. The method as claimed inclaim 9 wherein the electrochemical control has at least two electrodescontained on a plate with protrusions wherein the electrodes aredeposited on the protrusions of the plate and the plate is placed overthe microplate so that the protrusions of the plate fit into the wellsof the microplate.
 11. The method as claimed in claim 10 wherein theprotrusions are cone shaped.
 12. The method as claimed in claim 10wherein the protrusions are truncated cones.
 13. The method as claimedin claim 1 wherein the electrochemical control is operated to controlthe redox environment and detection scheme is operated subsequently orsimultaneously to detect changes in activity or binding in the sample.14. A system for performing analysis of a sample comprising: a testregion for the sample; an electrochemical control for the redoxenvironment of the sample, and a detection scheme selected from at leastone of the following: electrochemical; spectroscopic; radioassay; ormagnetic field measurement.
 15. The system as claimed in claim 14wherein the electrochemical control has at least two electrodes.
 16. Thesystem as claimed in claim 14 wherein the electrochemical control hasthree electrodes wherein the first electrode is an auxiliary electrode,the second electrode is a reference electrode, and the third electrodeis a working electrode.
 17. The system as claimed in claim 14 whereinthe electrochemical detection shceme comprises amperometry, voltammetry,capacitance, or impedance.
 18. The system as claimed in claim 14 whereinthe spectroscopic detection scheme comprises fluorescence, absorbance,infra red, phosphorescence, chemiluminescence, electroluminescence,Raman, electron spin resonance, or refractive index.
 19. The system asclaimed in claim 14 wherein a mediator titrant is added to the sample.20. The system as claimed in claim 14 wherein the electrochemicalcontrol is provided to control each of a multiplicity of test regions.21. The system as claimed in claim 20 wherein the test regions are eachof a multiplicity of wells in a microplate.
 22. The system as claimed inclaim 21 wherein the electrochemical control may be operated toseparately control the redox environment of each well in a microplate.23. The system as claimed in claim 22 wherein the electrochemicalcontrol has at least two electrodes contained on a plate withprotrusions wherein the electrodes are deposited on the protrusions ofthe top plate and the plate is placed over the microplate so that theprotrusions of the plate fit into the wells of the microplate.
 24. Thesystem as claimed in claim 23 wherein the protrusions are cone shaped.25. The system as claimed in claim 23 wherein the protrusions aretruncated cones.
 26. The system as claimed in claim 14 wherein theelectrochemical control is operated to control the redox environment andthe detection scheme is operated subsequently or simultaneously todetect changes in activity or binding in the sample.
 27. A method ofhigh throughput drug screening comprising: providing a surfacecomprising a plurality of test regions; providing an electrochemicalcontrol for the redox environment in each test region; adding at leastone target molecule or species to each of the test regions; adding atleast one sample containing a species to be tested to each test region;operating the electrochemical control for the redox environment of eachtest region separately; and analyzing the sample to identify ifinteraction has occurred between the target and the sample using adetection scheme selected from at least one of the following:electrochemical; spectroscopic; radioassay; or magnetic fieldmeasurement.
 28. The method as claimed in claim 27 wherein theelectrochemical control has at least two electrodes.
 29. The method asclaimed in claim 27 wherein the electrochemical control has threeelectrodes wherein the first electrode is an auxiliary electrode, thesecond electrode is a reference electrode, and the third electrode is aworking electrode.
 30. The method as claimed in claim 27 wherein theelectrochemical detection scheme comprises amperometry, voltammetry,capacitance, or impedance.
 31. The method as claimed in claim 27 whereinthe spectroscopic detection scheme comprises fluorescence, absorbance,infra red, phosphorescence, chemiluminescence, electroluminescence,Raman, electron spin resonance, or refractive index.
 32. The method asclaimed in claim 27 wherein a mediator titrant is added to the sample.33. The method as claimed in claim 27 wherein the test regions are amultiplicity of wells in a microplate.
 34. The method as claimed inclaim 33 wherein the electrochemical control has at least two electrodescontained on a plate with protrusions wherein the electrodes aredeposited on the protrusions of a plate and the plate is placed over themicroplate so that the protrusions of the plate fit into the wells ofthe microplate.
 35. The method as claimed in claim 34 wherein theprotrusions are cone shaped.
 36. The method as claimed in claim 34wherein the protrusions are truncated cones.
 37. A method for performinga non-biological high throughput assay comprising: providing a surfacecomprising a plurality of test regions; providing an electrochemicalcontrol for the redox environment in each test region; adding a sampleto each test region; operating the electrochemical control to controlthe redox environment of each test region separately; and analyzing thesample using a detection scheme selected from at least on of thefollowing: electrochemical; spectroscopic; radioassay; or magnetic fieldmeasurement;
 38. The method as claimed in claim 37 wherein theelectrochemical control has at least two electrodes.
 39. The method asclaimed in claim 37 wherein the electrochemical control has threeelectrodes wherein the first electrode is an auxiliary electrode, thesecond electrode is a reference electrode, and the third electrode is aworking electrode.
 40. The method as claimed in claim 37 wherein theelectrochemical detection scheme comprises amperometry, voltammetry,capacitance, or impedance.
 41. The method as claimed in claim 37 whereinthe spectroscopic detection method comprises fluorescence, absorbance,infra red, phosphorescence, chemiluminescence, electroluminescence,Raman, electron spin resonance, or refractive index.
 42. The method asclaimed in claim 37 wherein a mediator titrant is added to the sample.43. The method as claimed in claim 37 wherein the test regions are amultiplicity of wells in a microplate.
 44. The method as claimed inclaim 43 wherein the electrochemical control has at least two electrodescontained on a plate with protrusions wherein the electrodes aredeposited on the protrusions of the plate and the plate is placed overthe microplate so that the protrusions of the plate fit into the wellsof the microplate.
 45. The method as claimed in claim 44 wherein theprotrusions are cone shaped.
 46. The method as claimed in claim 44wherein the protrusions are truncated cones.